A Race to Stop EHV-1. A virologist's collaboration with marine science researchers could block the horse virus.

Art Frampton & Lauren Singletary

Novel Receptor

How does EHV-1 invade a horse cell?

Frampton and graduate student Lauren Singletary have identified a novel receptor, MHC class I (MHCI) - a portion of a horse cell that the virus uses as an entrance. Found in human and animal cells, MHCI normally aids the immune response system. When EHV-1 enters a horse cell via MHCI, it is able to replicate, spread and cause disease. Clinical signs of infection, such as fever, coughing, nasal discharge and neurological disease can show up within 24 hours after virus entry, but typically the incubation period is four to six days.

In addition to identifying which receptor the virus uses to enter the cell, Singletary and Frampton are working to determine which viral molecules attach to the receptor to permit virus entry.

“The virus particles of EHV-1 contain 13 different glycoproteins on the surface. These are special sugar-protein molecules that specifically interact with receptors to allow entry into the cell. I am trying to figure out which glycoproteins are binding to our receptor,” Singletary says. “Once I can determine that, I want to go further to find out specifically which part of the protein is binding to which part of the receptor.”

Singletary uses an enzyme immunoassay, a biochemical technique that detects the presence of an antibody or an antigen in a sample. She coats laboratory plates with cells that possess the MHCI receptor as well as those that do not. Then, she coats the cells with glycoprotein D (gD), known
to initiate entry in many strains of herpes virus.

“If it does interact with the receptor, then it should stick to the cells that have MHCI and not to the other ones,”
she explains.

Fever, depression, watery nose, loss of appetite and swollen legs and abdomen are the first symptoms to appear. As the disease spreads, some horses experience incontinence and the inability to stand. Pregnant mares are very susceptible to the virus, which can easily invade the sensitive endometrium surrounding a fetus and deprive the unborn foal of oxygen resulting in a stillbirth or weak foal that dies within days of birth.

Spread by direct contact with nasal secretions of infected horses through shared feed, water, buckets, blankets, tack and equipment or from the hands, boots or clothes of handlers, the virus is triggered continually for the rest of the horse’s life. Recurrences strike most commonly when the horse has a compromised immune system or is stressed by excessive heat, long transport to race or show, or new stable or pasture mates.

With the number of cases increasing dramatically in recent years and the racing industry holding its breath, scientists like UNCW virologist Art Frampton are working tirelessly to develop an anti-viral drug. Previously developed vaccines created to prevent the infection have proved weak and short-lasting.

Frampton’s approach is novel and two fold: provide appropriate surveillance measures to detect an outbreak of EHV-1 in a horse stable or farm, and, if EHV-1 is confirmed, administer an anti-viral drug to limit the spread of the virus. While the current anti-viral drugs won’t prevent infection, they will stop the spread of the disease through the equine body.

Blocking the Virus

In developing their research, Frampton and his laboratory team have a superior advantage at UNCW. At their fingertips are unique compounds from marine microalgae and cyanobacteria, isolated and purified by UNCW Center for Marine Science (CMS) chemists. Available to all UNCW faculty and members of the outside scientific community through collaborative studies and interactions,this collection contains thousands of compounds isolated from photosynthetic and non-photosynthetic marine organisms.

“What we have here is a library of organisms that have never been examined by anybody in detail in terms of chemical constituents and their biological properties,” says Carl B. Brown Distinguished Professor of Marine Science Jeffrey Wright, a bioorganic chemist at CMS.

Thanks to this one-of-a-kind collaboration and resource opportunity, Frampton received 480 chemical fractions - compound mixtures - from CMS researchers that he and his undergraduate students tested for their potential effectiveness in blocking the life cycle of EHV-1. Of these, one was found to be the best compound because it blocks virus replication while remaining non-toxic to the cell. Frampton and his students will continue to study this compound on a basic cellular level to determine how it blocks the virus.

According to Wright, this novel compound is produced by a photosynthetic dinoflagellate, a type of microalgae found in the ocean. Though some dinoflagellate species are toxic, other types produce non-toxic compounds, which may be beneficial for treating disease.

This discovery is just one example of several notable bioactive compounds found in Wright’s lab, including potential antibacterial and anticancer agents.

“If we can generate drug-resistant viruses, we might be able to go in and sequence those and see where the mutations in the virus are occurring. That might clue us into where and how the drug is acting,” Frampton says.

If this marine compound works, it could stop EHV-1 from spreading past the respiratory tract into an infected horse’s neurological or reproductive system, where it can do much more damage.

Human Applications: Fighting Cancer

Frampton’s EHV-1 research could have another use: cancer treatment. The human herpes simplex virus (HSV) has been shown to kill human cancer cells, but serious complications, like encephalitis - a swelling of the brain - remain a concern. Frampton’s hypothesis is that EHV-1, if used as a localized treatment in surgery, could kill human brain tumor cells with few side effects. His research shows that in tissue culture, EHV-1 can efficiently infect, replicate in and lyse, or kill, human brain tumor cells.

“We are genetically engineering the virus so that it only latches onto and infects the tumor cells while sparing the normal brain tissue,” Frampton says.

Currently funded by the Grayson-Jockey Club Research Foundation, Frampton is seeking further funding to expand his EHV-1 study into the realm of cancer research.

This ongoing EHV-1 research project has provided opportunities for more than 24 undergraduates - honors and directed independent study students - to participate in world-class viral research. Undergraduates are intimately involved in every aspect of the EHV-1 projects, from experimental design, running assays, collecting and analyzing data to co-authoring results for publication in scientific journals.

Directed independent study students Brian Kurtz ’10 and Sean Kelly ’10 serve as first and third authors and graduate student Lauren Singletary (see sidebar) as second author of an article describing how the novel entry receptor MHCI was discovered. The article, “Equus caballus Major Histocompatibility Complex Class I is an Entry Receptor for Equine Herpesvirus Type 1,” was published in the Journal of Virology 84 (Sept. 2010): 9027-9034.